Influenza A virus (IAV) is a major human respiratory pathogen that causes seasonal epidemics and occasional pandemics. The virus is a segmented, negative stranded RNA virus that, unlike most other RNA viruses, replicates its genome in the nucleus of infected cells. Genome replication yields a complementary RNA (cRNA) and a viral genomic RNA (vRNA). Both cRNA and vRNA contain a 5' triphosphate (ppp) which is recognized by the innate immune system by pattern recognition receptors (PRRs). Cytosolic sensors have been implicated in the specific recognition of influenza virus. Whether IAV can also be recognized in the nucleus is unknown. To address this, we screened nuclear proteins that, based on enzymatic features, may recognize viral RNA. Our preliminary results indicate that the protein DXO, a nuclear enzyme with 5'->3' pyrophosphohydrolase activity, can both recognize and degrade IAV RNA in vitro and in infected cells. We will study the mechanism and impact of DXO activity using biochemical and gene expression studies (Aims 1-2). We will examine DXO activity in vivo using a knock-out mouse model that we recently generated (Aim 3). These studies will provide novel mechanistic insights into how viral RNA expression is controlled along with elucidating the role of viral RNA catabolism in IAV pathogenesis, thus instructing new therapeutic avenues for disease eradication.